Diabetes mellitus reflects a metabolic disease, which can be found extensively all over the world. Persons with diabetes (PWD) have an impaired or missing production of the hormone insulin, which controls the blood-glucose level, and thus these persons bear a risk of hyperglycemia as well as hypoglycemia in case of inadequate insulin application [Definition and diagnosis of diabetes mellitus and intermediate hyperglycaemia. WHO, and IDF (2006); WHO Document Production Service ISBN 9241594934].
To ensure a correct application of insulin, highly specific, accurate and easy to handle glucose measurement systems are needed for both self-measurement systems and high-throughput measurement systems on clinical scale.
To allow the adequate determination of glucose concentrations in the blood and to make the measurement highly specific, enzymatic reactions are involved. These days, the two types of enzymes that are used in diabetes analytics are reflected by the glucose dehydrogenases (GDH(s)) and glucose oxidases (GOx(s)) [Hönes et al. (2008) Diabetes Technol. Therap. 10:10-26].
The main advantage of GDHs is their oxygen-independent oxidation of glucose, but these enzymes show slight side-activities on certain other clinical relevant sugars, and thus GDHs can be unspecific [Olsthoorn & Duine (1998) Biochem. 37:13854-13861]. By contrast, the GOxs are highly specific for glucose, but their oxidation is strongly oxygen-dependent [Bankar et al. (2009) Biotechnol. Advances 27:489-501; and Bentley & Neuberger (1949) Biochem. J 45:584-590].
In more detail, GOxs as flavoproteins belong to the family of oxidoreductases (i.e., β-D-glucose:oxygen 1-oxidoreductase). Native or wild-type (WT) GOxs catalyze an oxidation of β-D-glucose to D-glucono-δ-lactone and hydrogen peroxide (H2O) by employing molecular oxygen as an electron acceptor [see e.g., Pazur & Kleppe (1964) Biochem. 3:578-583]. The reaction is depicted by the following formula:D-glucose+oxygen→gluconolactone+H2O.
The substrates of the GOxs can be divided into two (2) groups: (i) the electron acceptors of the oxidative half reaction; and (ii) the electron donors of the reductive half reaction [see e.g., Leskovac et al. (2005) Int. J. Biochem. Cell Biol. 37:731-750]. One of skill in the art is aware that apart from D-glucose various derivatives of D-glucose are potential substrates for the reductive half reaction of GOxs.
GOxs from different origins have been described so far. For instance, the GOx from marine algae Chondrus crispus is described in U.S. Pat. Nos. 7,544,795 and 6,924,366; the GOx from filamentous fungi Cladosporium spec. is described in Intl Patent Application Publication Nos. WO 95/29996 and WO 1998/020136; and the GOx from Talaromyces flavus is described in U.S. Pat. No. 6,054,318.
The best-described GOx in literature is from Aspergillus niger [Hecht et al. (1993) Biosens. Bioelectron. 8:197-203; and Wohlfahrt et al. (1999) Acta Crystallographica Section D Biological Crystallography 55:969-977].
Int'l Patent Application Publication No. WO 89/126675 describes producing GOxs from A. niger in recombinant systems, and Int'l Patent Application Publication No. WO 2008/079227 describes a GOx obtained from A. niger formulated in a composition conferring improved storage stability.
GOx is a well-characterized protein forming a dimer of 160 kDa in size, and crystal structures have been solved thereof [Hecht et al. (1993) J. Mol. Biol. 229:153-172].
It is further known that particularly the wild-type GOx of A. niger (GOx-WT) exhibits significant temperature stability and specificity for the substrate glucose. Additionally, GOx is a glycoprotein with a high-mannose type carbohydrate content of 10%-16% [Hayashi & Nakamura (1981) Biochim. Biophys. Acta 657:40-51; and Pazur et al. (1965) Arch Biochem. Biophys. 111:351-357].
These days, GOxs are commonly used in test elements such as biosensors for detecting glucose either in industrial solutions or in bodily fluids of a subject (e.g., in blood and urine).
Many currently available self-measurement devices are electrochemical biosensors consisting in principle of (a) a biological component (i.e., the respective enzyme having glucose as substrate); (b) an indicator (the electronic component); and (c) a signal transducer.
In the measurement device, electrons from the glucose are transferred by the biological component (a) to an electrode via mediators (b). A signal transducer (c) then converts the electrical signal into a real-time glucose concentration, which is proportional to the amount of transferred electrons.
Apart from the above-described electrochemical sensors, photometric sensors also are available. The difference here is that the electrons from the glucose are transferred to redox-indicator dye (serving as indicator). The resulting color change of the reduced dye is measured photometrically.
Another main application might be using GOxs in the anodic compartment of implanted and miniaturized biofuel cells burning glucose from the blood stream and thereby powering miniature diagnostic devices or pumps.
Moreover, GOx applications in the food industry are numerous, since its capability of generating H2O2, which has an anti-microbial effect, and can be utilized to improve the storage stability of certain food products including cheese, butter and fruit juice.
Applications of GOxs in cosmetic compositions may utilize the anti-microbial properties as well. Potential uses for hexose oxidases in pharmaceutical and cosmetic compositions were suggested in U.S. Pat. Nos. 6,924,366 and 6,251,626, as well as Int'l Patent Application Publication No. WO 2007/045251.
Furthermore, GOx can be used to produce transgenic plants and other organisms with reduced susceptibility or increased resistance to pests or diseases (see e.g., Int'l Patent Application Publication No. WO 1995/021924).
However, use of GOxs in glucose biosensors is of significant interest in accordance with this disclosure. In this regard, Int'l Patent Application Publication No. WO 2009/104836 describes a glucose biosensor including a genetically engineered GOx variant improved for attaching to metal surfaces.
GOx mutants derived from A. niger are known and are mutated in T30V and/or I94V. Likewise, the corresponding double-mutant T30V; I94V is known [Zhu et al. (2006) Biosens. Bioelectron. 21:2046-2051; and Zhu et al. (2007) Biotechnol. J. 2:241-248].
Specifically, the above-mentioned T30V and I94V double-mutant exhibits slightly increased enzyme activity (kcat from 69.5/s to 137.7/s), exhibits an increased thermostability in a range of 58° C. to 62° C., and exhibits an improved pH stability in a range of 8 to 11 when compared to GOx-WT. However, the double-mutant derived from A. niger exhibits equal oxygen consumption rates when compared to the GOx-WT.
EP Patent Application Publication No. 3 415 863 describes nucleic acid molecules and polypeptides thereof having GOx activity but being mutated in at least three (3) of the following amino acid positions: 2, 13, 30, 94 and 152. Particularly, the M12 variant having the substitutions N2Y, K13E, T30V, I94V and K152R shows, besides an increased expression level in S. cerevisiae, a twice-increased activity for oxygen as electron acceptor.
Horaguchi et al. identified one amino acid residue position being involved in the oxidative half reaction of the GOx variants described therein, being it the GOx of Penicillium amagasakiense and the GOx variant of A. niger [Horaguchi et al. (2012) Meet. Abstr. MA2012-02 18:2057]. The one position is S114 of the P. amagasakiense GOx variant, and the corresponding T110 of the A. niger variant. Both positions were replaced by the amino acid Ala leading to a decrease in activity for oxygen as electron acceptor. For instance, the GOx variant of A. niger exhibited a 6.6-fold reduced oxygen consumption, and thus had a residual oxygen activity of 30.4% besides a mediator activity of 363%.
One drawback of GDHs is that they generally are unspecific for glucose, and the oxygen-dependent GOxs represent the key enzymes of current glucose measurement systems.
The solution to the underlying problem is providing specifically modified and thus optimized GOx variants derived from A. niger. 
Surprisingly and unexpectedly, this disclosure provides novel GOx variants derived from A. niger that are specific for glucose, but independent from oxygen for glucose oxidation and thus more accurate for glucose measurements. Moreover, this disclosure provides novel GOx variants that are specific for glucose, and thereby exhibit significantly reduced oxygen consumption rates and/or significantly increased mediator activity for electron mediators other than oxygen.
The GOx variants herein, having besides the two substitutions T30V and I94V according to SEQ ID NO:1, additionally at least one amino acid substitution in any of the six (6) positions selected from: S53; A137; A173; A332; F414 and V560.
The optimized GOx variants herein are suitable to be implemented in improved blood-glucose measurement systems.